Date of Award

6-2015

Degree Name

Master of Science

Department

Biological Sciences

First Advisor

Dr. Don A. Kane

Second Advisor

Dr. Pamela Hoppe

Third Advisor

Dr. Todd Barkman

Keywords

Cyclin B1, Zebrafrsh, Apoptosis, Cell Cycle, Early development

Access Setting

Masters Thesis-Open Access

Abstract

Cell division is controlled by genes that regulate the cell cycle. Here we show that the zebrafish specter (spr) mutant is mutation in the cyclin B1 gene, a gene necessary for the G2 to M transition of the cell cycle.

The spr mutation mapped to the cyclin B1 gene. Sequencing showed a transition (C139→T) that caused a nonsense mutation in exon 2 of the cyclin B1 gene. In situ hybridization of cyclin B1 revealed that the mRNA is absent in the mutant embryo by gastrulation. We found that the earliest visible mutant phenotype was a darkening of the head caused by the appearance of apoptotic cells in the brain. In situ hybridization of markers expressed in neural precursors and blood cells showed that, in general, mutant embryos have bigger and fewer cells. In situ hybridization of notch1b and deltaa also showed fewer neural stem cells that give rise to fewer but bigger neuronal precursors.

Phospho-histone-3 antibody staining suggested that the cell cycle is delayed in the mutant embryo. The expression of the Dual FUCCI transgene in the live spr mutant embryos confirmed that cells are delayed in the S/G2/early M phase of the cell cycle.

CRISPR/Cas9 mediated germline mutagenesis of the wild-type cyclin B1 allele showed that the spr mutant phenotype is a result of a nonfunctional Cyclin B1 gene. We conclude that the spr mutant phenotype is caused by the mutation in the cell cycle gene cyclin B1, an essential regulator of the cell cycle progression from the G2 to M phase of the cell cycle. This then leads to mitotic abnormalities and finally developmental arrest.

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